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HFV Ebola sequence database

[Abstract #532]

World-wide Evaluation of DNA Sequencing Approaches for the Identification Drug Resistance Mutations in the HIV-1 Reverse Transcriptase

R. SCHUURMAN, L. DEMETER, P. REICHELDERFER, J. TIJNAGEL, T. DE GROOT,C. BOUCHER on behalf of the ENVA laboratories, the Sequencing Working Group and participating laboratories.

Presented at the 5th Conference on Retroviruses and Opportunistic Infections, Chicago, IL, February 1998.


Objective: To evaluate the accuracy and sensitivity of applied methods and technologies for determination of drug resistance mutations in the HIV-1 RT gene.

Methods: The ENVA genotyping panel, consisting of a blinded set of 9 low concentration plasmid mixtures containing HIV-1 RT genes at different rations of wildtype and mutant genotypes at amino acid positions 41 (ATG/CTG), 184 (ATG/ATA/GTG), 215 (ACC/TAC), was distributed among 23 established "HIV-resistance" laboratories in Europe and United States. Participants amplified and genotypically characterized the samples using their standard laboratory methods. For each of the identified heterogenic positions proportion of wild type and mutant genotype were reported.

Results: Automated sequencing technologies were applied by all of the labs: 17 labs used Applied Biosystems equipment, 3 used Pharmacia technology 1 used a Vistra and 2 used Affymetrix technology. All labs reported the presence of sequence heterogeneities at codons 4! 1, 215, 184 in one or more of the panel samples, though not all reported the correct (wild type) codons. A few labs reported sequence heterogeneities at non-variable positions. The presence of 25% mutant was detected by 13 labs for codon 41 labs for codon 215, 4 labs for codon 184 lle and 16 labs for codon 184 Val, and the reported fractions generally ranged between 10 and 70%. In panel samples containing 50% mixture of wild type and mutant genotype the mutant was detected within 10% accuracy by 14 of 23 labs for 184 lle, 12 for 184 Val, 9 labs for 41 and 14 for codon 2 Reported results which were more than 10% off of the 50% input concentration ranged between 0 and 100%.

Conclusion: This first multicenter evaluation of methods for the genotype identification HIV-1 drug resistance mutations in the RT gene demonstrates that large differences in the sensitivity and accuracy of DNA sequence analysis approaches. We conclude that proper quality assurance is urgently needed.

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